Therapeutic Uses of Glandular Kallikrein

ABSTRACT

Provided is an immunosuppressive peptide of 40 kDa molecular weight, isolated from the submandibular glands (SMG*) of rats, having the capacity to suppress immune reactions upon parenteral administration to rats and mice. The peptide was identified as glandular kallikrein (K1) by partial sequencing and by enzymatic activity. Also provided are methods and compositions for Using K1 peptides in the treatment of autoimmune diseases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/705,284 filed Feb. 12, 2007, which is a continuation of U.S.application Ser. No. 10/162,697 filed Jun. 6, 2002, now U.S. Pat. No.7,195,759, which claims priority under 35 USC §119(e) to U.S.Provisional Application No. 60/296,153 filed Jun. 7, 2001; each of whichis incorporated by reference in its entirety.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is DIAM_(—)015_(—)04US_ST25.txt. The text file is 4KB, was created on Aug. 21, 2012, and is being submitted electronicallyvia EFS-Web.

BACKGROUND OF THE INVENTION

Kallikreins belong to a family of serine proteases capable of cleavingvarious substrates and generating biologically active peptides. In spiteof the names, tissue or glandular kallikreins should be distinguishedfrom plasma kallikrein. They differ from plasma kallikrein in theirgenes of origin, molecular weight, amino acid sequences, substrates,peptide products and most probably physiological functions. There are atleast 20 genes for tissue kallikrein in rodents (98), while in humans 15genes have been so far described (Yousef, G. M., Scorilas, A., Jung, K.,Ashworth, L. K., Diamondis, E. P. J. Biol. Chem. 2001, 276:53-61;Clements, J., Hooper, J., Dong, Y., Harvey, T. Biol. Chem. 2001,382:5-14). Of these, only one gene in each species codes for trueglandular kallikrein. Of the rat genes at least 6-7 appear to beexpressed in the submandibular (SM) gland (99). These include trueglandular kallikrein, tonin, a and y nerve growth factor (NGF), and theepidermal growth factor (EGF)-binding protein (EGF-BP), type A, B, andC. As used herein, the term “glandular kallikrein (GK)” refers to trueGK, while the general term “kallikrein(s)” (K), will be used for anyunspecified members of the tissue kallikrein family. True GK has beendesignated in various species as kallikrein-1 (K 1) (100).

The best known substrates for GK-action are hepatic-derived kininogens(100) which occur in two forms: low molecular weight kininogen (50 kDa)and high molecular weight kininogen (120 kDa). From the action of plasmakallikrein on high molecular weight kininogen a nonapeptide, bradikinin,is generated, while in most species GK gives rise to a decapeptide,kallidin (lys-bradikinin) from either low or high molecular weightkininogen. Kallidin is biologically active in itself but may also befurther processed into bradikinin. An exception may be the GK of the ratSMG which was reported to produce bradikinin (101).

While the action of GK on kininogen is particularly well studied, thefull range of GK substrates has not yet been investigated. Since GK is,and most of the times remains, localized in certain tissues,physiological substrates are likely to vary from tissue to tissue. Ofparticular interest here is the possibility that GK may activate or inany way regulate some other immunologically active factors of the SMgland, including NGF, EGF/transforming growth factor (TGF)-α and TGFβ.Thus, salivary gland GK may exert its immunological effects either viathe production of classical kinins; or via other immunologically activefactors. Moreover, salivary GK is actively secreted in saliva (102) andwould be expected to reach various points in the gastro-intestinal tractand act on various substrates there.

SUMMARY OF THE INVENTION

Certain embodiments of the present invention relate to methods ofsuppressing an immune reaction to beta cells in a human afflicted withor exhibiting at least one clinical sign of type 1 diabetes, comprisingadministering to the human about 10 to about 5,000 International Unitsof a glandular kallikrein, and thereby suppressing the immune reactionto beta cells.

In some embodiments, the glandular kallikrein comprises kallikrein-1, afragment of a kallikrein having serine protease activity, or a mixturethereof. In particular embodiments, the glandular kallikrein isadministered orally. In specific embodiments, the glandular kallikreinis administered parenterally.

Some embodiments include administering the glandular kallikrein and abeta cell antigen. Particular embodiments include co-administering thebeta cell antigen with the glandular kallikrein. In some embodiments,the glandular kallikrein and the beta cell antigen are administeredorally.

According to one aspect of the invention, there is provided apharmaceutical composition comprising glandular kallikrein or abioactive fragment thereof and an antigen.

The antigen may be an auto-antigen. The auto-antigen may be from anautoimmune disease selected from the group consisting of: rheumatoidarthritis, lupus erythrematosis, multiple sclerosis, inflammatory boweldiseases, for example, Crohn's disease and ulcerative colitis,autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura,autoimmune hepatitis and pancreatitis, Goodpasture's syndrome, acuterheumatic fever, pemphigus vulgaris, myasthenia gravis, ankylosingspondylitis, acute anterior uveitis, Grave's disease, Hashimoto'sthyroiditis and juvenile diabetes.

The antigen may be a gene therapy vector.

According to a second aspect of the invention, there is provided amethod of inducing tolerance to at least one orally administered antigenin an animal comprising administering to said animal an effective amountof a glandular kallikrein or a bioactive fragment thereof.

The amount administered may be sufficient to reduce the animal's immuneresponse to the antigen.

The method may include co-administering an antigen with the glandularkallikrein.

The antigen may be from an autoimmune disease selected from the groupconsisting of: rheumatoid arthritis, lupus erythrematosis, multiplesclerosis, inflammatory bowel diseases, for example, Crohn's disease andulcerative colitis, autoimmune hemolytic anemia, autoimmunethrombocytopenic purpura, autoimmune hepatitis and pancreatitis,Goodpasture's syndrome, acute rheumatic fever, pemphigus vulgaris,myasthenia gravis, ankylosing spondylitis, acute anterior uveitis,Grave's disease, Hashimoto's thyroiditis and juvenile diabetes.

According to a third aspect of the invention, there is provided a methodof ameliorating symptoms associated with an autoimmune disorder in ananimal afflicted with said autoimmune disorder comprising administeringto said animal an effective amount of a glandular kallikrein or abioactive fragment thereof.

The method may include co-administering an antigen with the glandularkallikrein.

The autoimmune disease may be selected from the group consisting of:rheumatoid arthritis, lupus erythrematosis, multiple sclerosis,inflammatory bowel diseases, for example, Crohn's disease and ulcerativecolitis, autoimmune hemolytic anemia, autoimmune thrombocytopenicpurpura, autoimmune hepatitis and pancreatitis, Goodpasture's syndrome,acute rheumatic fever, pemphigus vulgaris, myasthenia gravis, ankylosingspondylitis, acute anterior uveitis, Grave's disease, Hashimoto'sthyroiditis and juvenile diabetes.

According to a fourth aspect of the invention, there is provided amethod of enhancing an immune response to at least one orallyadministered antigen comprising administering to said animal aneffective amount of a glandular kallikrein inhibitor.

The amount administered may be sufficient to reduce the animal'stolerance to the antigen.

The method may include co-administering an antigen with the glandularkallikrein inhibitor.

The antigen may be an oral vaccine.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows glandular kallikrein inhibition of the effector phase ofcontact sensitivity in mice. A/J mice were sensitized by application of0.1 ml of a 5% solution of picryl chloride in ethanol to the clippedskin of the abdomen. On day five after sensitization the mice receivedaprotinin (Apr.), injected s.c. as a full dose (190 μg/animal) or as ahalf dose. Fifteen minutes later the animals received a further s.c.injection of rat (r) or pig (p) glandular kallikrein (GK) (57 μg/animal;corresponding to the yield from one SMG) in 0.2 ml of PBS. Controlsreceived PBS alone. On day 6 the mice were challenged by the applicationof 0.1% solution of picryl chloride in olive oil to both sides of theear. The thickness of the ear was measured 24 hrs after challenge andthe results were compared with that of controls. The results areexpressed in units of 1/10 mm. The bars represent the mean increase ofthickness±SE. of the challenged ear. Unimmunized controls did not showany increase in ear thickness (not shown).

FIG. 2A shows the effect of SMGx and rK1 on CA and on the induction oforal tolerance in Sprague Dawley rats. Compared to control: a) p<0.05 b)p<0.01 c) p<0.001 Compared to tolerized group: 1) p<0.05 2) p<0.01 3)p<0.001 Groups of 4 male Sprague-Dawley rats were used, weighing 200-300g. SMGx was performed on day −21, treatment with native bovine type IIcollagen was done with 3 μg of daily dose by gavage on days −7, −5 and−2 Immunization was done with 75 μg of BCII in incomplete Freund'sadjuvant, injected i.d. at the base of the tail on days 0 and on 7.Semipurified rK1 (GK) was given s.c. in doses equivalent to 50% of theyield from one SMG rat on days 16, 18 and 20.

FIG. 2B-Analysis of variance report, day 22. Bonferroni (with control)multiple-comparison test Response: 1, 2, 3, 4, 5.

Term A:

Group Count Mean Different from groups Alpha = 0.050 Error term = S(A)DF = 45 MSE = 1.653333E−02 Critical Value = 2.602083 2 lmm + Tol 10 3.73, 4, 5, 1 3 lmm + SMGx 10 3.98 2, 1 4 lmm + Tol + SMGx 10 4.04 2, 1 5lmm + SMGextract 10 4.04 2, 1 1 Immunized 10 4.3 2, 3, 4, 5 Alpha =0.010 Error Term = S(A) DF = 45 MSE = 1.653333E−02 Critical value3.202842 2 lmm + Tol 10 3.7 3, 4, 5, 1 3 lmm + SMGx 10 3.98 2, 1 4 lmm +Tol + SMGx 10 4.04 2, 1 5 lmm + SMGextract 10 4.04 2, 1 1 Immunized 104.3 2, 3, 4, 5 Alpha = 0.001 Error Term = S(A) DF = 45 MSE =1.653333E−02 Critical Value 3.977715 2 lmm + Tol 10 3.7 3, 4, 5, 1 3lmm + SMGx 10 3.98 2, 1 4 lmm + Tol + SMGx 10 4.04 2, 1 5 lmm +SMGextract 10 4.04 2, 1 1 Immunized 10 4.3 2, 3, 4, 5

FIG. 3A shows that salivary K1 is required for the induction of oraltolerance against CA in Lewis rats. Compared to control: a) p<0.05 b)p<0.01 c) p<0.001 Compared to tolerized group: 1) p<0.05 2) p<0.01 3)p<0.001 Groups of 5 female Lewis rats were used. SMGx was performed onday −21. Kallikrein was given at doses of 100 IU/0.5 ml/rat by gavage ondays −8, −5, −3. Native bovine collagen type II (Sigma) was given orallyat doses of 3 μg/rat in 0.5 ml distilled water on days −7, −5, −3 (39).Immunization was done on days 0 and day 7 with BCII dissolved in 1 mMacetic acid (at 1.5 mg/ml) and emulsified with an equal volume ofincomplete Freund's adjuvant. Each rat received 0.1 ml of antigen(containing 75 μg BCII) injected interdermally at the base of the tail(8). The diameters of the hind paws were measured, and plotted in thefigure as mean±SE.

FIG. 3B. Analysis of variance report, day 25 Bonferroni (with control)multiple-comparison test Response: 1, 2, 3, 4, 5.

Term A:

Group Count Mean Different from groups Alpha = 0.050 Error term = S(A)DF = 33 MSE = 0.5016446 Critical Value = 2.642069 5 Imm, Tol SmGx, pGK 84.5 3, 4, 1 2 lmm + Tot 8 4.575 3, 4, 1 3 lmm + SMGx 8 5.55625 5, 2 4lmm + Tol + SMGx 8 5.8875 5, 2 1 Immunized 8 6.566667 5, 2 Alpha = 0.010Error Term = S(A) DF = 33 MSE = 0.5016446 Critical value 3.272916 5 Imm,Tol SmGx, pGK 8 4.5 4, 1 2 lmm + Tol 8 4.575 4, 1 3 lmm + SMGx 8 5.556254 lmm + Tol + SMGx 8 5.8875 5, 2 1 Immunized 8 6.566667 5, 2 Alpha =0.001 Error Term = S(A) DF = 45 MSE = 1.653333E−02 Critical Value3.977715 5 Imm, Tol SmGx, pGK 8 4.5 1 2 Imm + Tol 8 4.575 1 3 Imm + SMGx8 5.55625 4 lmm + Tol + SMGx 8 5.8875 1 Immunized 8 6.566667 5, 2

FIG. 4 shows the effects of pooled Sephacryl fractions of submandibularand parotid glands on the Con A stimulated lymph node cellproliferation. The effects of pooled Sephacryl fractions ofsubmandibular (, ▪) and parotid (◯, □) glands on the Con A stimulatedlymph node cell proliferation: (, ◯) MW>50 kDa; (, □) MW<50 kDa. Thesolid horizontal line is the value of mean c.p.m. for the controls andthe shaded area represents the 95% confidence limits. From Ref. 12.

FIG. 5 is an SDS PAGE of the purified protein from rat SM gland (lane2). Molecular weight standards are shown in lane 1.

FIG. 6 is a partial N-terminal amino acid sequence of the 40 kDa protein(SEQ ID NO:1) isolated from rat SM gland compared with those of membersof kallikrein family expressed in the rat SM gland (SEQ ID Nos. 2-8).The boxed areas represent regions of identity with the 40 kDa protein.Blank spaces are used to align homologous sequences in differentproteins. x=not identified.

FIG. 7 shows increased proliferative activity of Con A stimulated A/Jlymph node cells induced by rGK and pGK and reversal of this effect withaprotinin. The rGK and pGK doses were 1.78 μg/culture (finalconcentration 0.22 μM/l). Aprotinin (Apr.) doses were: 6 μg, 3 μg, or1.5 μg per culture 4.6 μM/l, 2.3 μM/l, and 1.15 μM/l, respectively).Results are expressed as counts per minute (C.P.M.) in triplicatecultures (S.D.).

FIG. 8 shows the effects of single or multiple injections of asemi-purified preparation of rGK in the CA model. Effects of a single(▾, day 14) or multiple (▪, days 14, 18 and 24) injections of asemi-purified preparation of rGK in the CA model. Controls () receivedPBS only.

TABLE 1 shows functional disability in rats with arthritis.

TABLE 2 shows effects of high molecular weight (HMW) and low molecular(LMVV) pools of gel filtration fractions in three in viva immunologicalassays.

TABLE 3 shows effects of the intradermal injection of rGK given beforeor after immunization in the DTH model.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned hereunderare incorporated herein by reference.

As used herein, “effective amount” refers to the administration of anamount of a given compound that achieves the desired effect.

As used herein, “purified” does not require absolute purity but isinstead intended as a relative definition. For example, purification ofstarting material or natural material to at least one order ofmagnitude, preferably two or three orders of magnitude is expresslycontemplated as falling within the definition of “purified”.

As used herein, “glandular kallikrein” refers to the enzymes describedin different mammalian species by several authors (Richards, R. I.,Catanzaro, D. F., Mason, A. J., Morris, B. J., Baxter, J. D., Shine, J.,J. Biol. Chem. 1982, 257:2758-61; James, M. N., Delbaere, L. T., Brayer,G. D., Can. J. Biochem. 1978, 396-402; Fiedler, F. Lemon, M. J.,Hirschauer, C., Leysath, G., Lottspech, F., Henschen, A., Gau, W.,Bhoola, K. D., Biochem. J. 1983, 125-34; Swift, G. H., Dagorn, J. C.,Ashley, P. L., Cummings, S. W., MacDonald, R. J., Proc. Nat. Acad. Sci.U.S.A., 1982, 79: 7263-7; Tschetsche, H., Mair, G., Godec, G., Adv. Exp.Med. Biol. 1979, 120A:245-60). We are not making any claims here aboutpossible uses of other members of the serine protease family.

As used herein, the term “treating” in its various grammatical formsrefers to preventing, curing, reversing, attenuating, alleviating,minimizing, suppressing or halting the deleterious effects of a diseasestate, disease progression, disease causitive agent or other abnormalcondition.

As used herein, “autoimmune disease” refers to diseases wherein the hostimmune system recognizes “self” material as foreign. Examples includebut are by no means limited to rheumatoid arthritis, multiple sclerosis,inflammatory bowel diseases, for example, Crohn's disease and ulcerativecolitis, autoimmune hemolytic anemia, autoimmune thrombocytopenicpurpura, autoimmune hepatitis and pancreatitis, Goodpasture's syndrome,acute rheumatic fever, pemphigus vulgaris, myasthenia gravis, ankylosingspondylitis, acute anterior uveitis, Grave's disease, Hashimoto'sthyroiditis and juvenile diabetes.

As used herein, “antigen” refers to any material potentially recognizedby a host immune system as “non-self”.

As used herein, “a biologically active fragment” refers to a fragment ofthe glandular kallikrein, which retains its biological activity, thatis, serine protease activity.

As used herein, “animal” refers to vertebrates. It is of note that thepatient may be a human.

Described herein is a method of enhancing tolerance in an animal tomaterial recognized by the animal as foreign comprising administeringglandular kallikrein either in combination with or prior to hostexposure to the foreign material. In some embodiments, the “foreign”material, for example an antigen, and the glandular kallikrein are takenorally. It is of note that the “foreign” material may be for example anauto-antigen.

In some embodiments, the glandular kallikrein, or a bioactive fragmentthereof, with or without an antigen may be combined with other compoundsor compositions known in the art such that the glandular kallikrein is apharmaceutical composition in the form of, for example, a pill, tablet,liquid, film or coating using means known in the art and as discussedbelow.

It is of note that the glandular kallikrein with or without an antigendiscussed above may be prepared to be administered in a variety of ways,for example, topically, orally, intravenously, intramuscularly,subcutaneously, intraperitoneally, intranasally or by local or systemicintravascular infusion using means known in the art and as discussedbelow.

It is of note that as discussed herein, the above-describedpharmaceutical composition may be arranged to be delivered in 3-dayintervals at an oral dosage of about 10-5000 International Units, or ofabout 100-1000 IU, or more preferably, 500 International Units (IU) perkg of the subject. This dosage is based on our experience with rats.This in other embodiments, the daily dosage may be about 170 IU/kg ofthe subject. As will be apparent to one knowledgeable in the art, thetotal dosage will vary according to the weight, health and circumstancesof the individual.

In some embodiments, the above-described pharmaceutical composition atconcentrations or dosages discussed above may be combined with apharmaceutically or pharmacologically acceptable carrier, excipient ordiluent, either biodegradable or non-biodegradable. Exemplary examplesof carriers include, but are by no means limited to, for example,poly(ethylene-vinyl acetate), copolymers of lactic acid and glycolicacid, poly(lactic acid), gelatin, collagen matrices, polysaccharides,poly(D,L lactide), poly(malic acid), poly(caprolactone), celluloses,albumin, starch, casein, dextran, polyesters, ethanol, mathacrylate,polyurethane, polyethylene, vinyl polymers, glycols, mixtures thereofand the like. Standard excipients include gelatin, casein, lecithin, gumacacia, cholesterol, tragacanth, stearic acid, benzalkonium chloride,calcium stearate, glyceryl monostearate, cetostearyl alcohol,cetomacrogol emulsifying wax, sorbitan esters, polyoxyethylene alkylethers, polyoxyethylene castor oil derivatives, polyoxyethylene sorbitanfatty acid esters, polyethylene glycols, polyoxyethylene stearates,colloidol silicon dioxide, phosphates, sodium dodecylsulfate,carboxymethylcellulose calcium, carboxymethylcellulose sodium,methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose,hydroxypropylmethycellulose phthalate, noncrystalline cellulose,magnesium aluminum silicate, triethanolamine, polyvinyl alcohol,polyvinylpyrrolidone, sugars and starches. See, for example, Remington:The Science and Practice of Pharmacy, 1995, Gennaro ed.

As will be apparent to one knowledgeable in the art, specific carriersand carrier combinations known in the art may be selected based on theirproperties and release characteristics in view of the intended use.Specifically, the carrier may be pH-sensitive, thermo-sensitive,thermo-gelling, arranged for sustained release or a quick burst. In someembodiments, carriers of different classes may be used in combinationfor multiple effects, for example, a quick burst followed by sustainedrelease.

In other embodiments, the above-described pharmaceutical composition atconcentrations or dosages described above may be encapsulated fordelivery. Specifically, the pharmaceutical composition may beencapsulated in biodegradable microspheres, microcapsules,microparticles, or nanospheres. The delivery vehicles may be composedof, for example, hyaluronic acid, polyethylene glycol, poly(lacticacid), gelatin, poly(E-caprolactone), or a poly(lactic-glycolic) acidpolymer. Combinations may also be used, as, for example, gelatinnanospheres may be coated with a polymer of poly(lactic-glycolic) acid.As will be apparent to one knowledgeable in the art, these and othersuitable delivery vehicles may be prepared according to protocols knownin the art and utilized for delivery of the glandular kallikrein.Alternatively, the delivery vehicle may be suspended in saline and usedas a nanospray for aerosol dispersion onto an area of interest.Furthermore, the delivery vehicle may be dispersed in a gel or paste,thereby forming a nanopaste for coating a tissue or tissue portion.

It is of note that the glandular kallikrein compositions as describedabove may be combined with permeation enhancers known in the art forimproving delivery. Examples of permeation enhancers include, but are byno means limited to those compounds described in U.S. Pat. Nos.3,472,931; 3,527,864; 3,896,238; 3,903,256; 3,952,099; 4,046,886;4,130,643; 4,130,667; 4,299,826; 4,335,115; 4,343,798; 4,379,454;4,405,616; 4,746,515; 4,788,062; 4,820,720; 4,863,738; 4,863,970; and5,378,730; British Pat. No. 1,011,949; and Idson, 1975, J. Pharm. Sci.64:901-924.

In some embodiments, the pharmaceutical composition in any suitable formas described above, may be combined with biological or synthetictargeting molecules, for example, site-specific binding proteins,antibodies, lectins or ligands, for targeting the glandular kallikreinto a specific region or location.

Described below are sample treatments for exemplary autoimmune andhyper-sensitive disorders for illustrative purposes. As will be apparentto one of skill in the art, the treatment methods may be applied to anysuitable disease or disorder. It is further of note that antigensimplicated in many autoimmune disorders are well known in the art.

As discussed above, multiple sclerosis is a chronic neurologicaldisorder that affects the nervous system and is believed to be anautoimmune disorder. Specifically, cell migration of a macrophage-likeactivity is involved in the destruction of the myelin. As discussedabove, administration of glandular kallikrein or a bioactive fragmentthereof combined with antigen(s) related to multiple sclerosis orpossibly the myelin itself would induce tolerance of these compounds,thereby inhibiting demyelination by the patient's immune system, therebyreducing severity of the disease. That is, the glandular kallikreincomposition would accomplish at least the following: decrease theseverity of symptoms, decrease the duration of disease exacerbations,increase the frequency and duration of disease remission and/or symptomfree periods, prevent or attenuate chronic progression of the disease,improve visual symptoms, improve gait disorders, such as, weakness,axial instability, sensory loss, spasticity, hyperreflexia and/or lossof dexterity, improve cognitive impairment, reduce myelin loss, reducebreakdown of the blood-brain barrier and reduce perivascularinfiltration of mononuclear cells. In these embodiments, the glandularkallikrein composition may be ingested as a tablet or pill, appliedtopically or injected, prepared at appropriate concentrations or dosagesas described herein.

Similarly, inflammatory bowel diseases are caused by intestinalinflammation and repeated inflammatory responses. As discussed above,administering glandular kallikrein or a bioactive fragment thereof withantigen(s) implicated in or related to the inflammatory bowel diseaseswould induce tolerance, thereby reducing the severity of the disease.Specifically, the glandular kallikrein composition would accomplish atleast one of the following: decrease the frequency of the attacks,increase the duration of remission periods, decrease the severity orduration of abscess formation, intestinal obstruction, intestinalperforation and the like as well as ameliorate or reduce symptoms suchas bloody diarrhea, abdominal pain, fever, weight loss and abdominaldistension.

Arthritis is believed to be an autoimmune disease, characterized byinfiltration of the joints with inflammatory system cells. As such,administration of glandular kallikrein or a bioactive fragment thereofin combination with antigen(s) related to or implicated in arthritiswill induce tolerance to these antigens, thereby inhibiting progressionof the autoimmune disease. Specifically, the glandular kallikreincomposition will accomplish at least one of the following: decreaseseverity of symptoms, including pain, swelling and tenderness ofaffected joints, weakness and fatigue, decrease severity of clinicalsigns, including thickening of the joint capsule, synovial hypertrophy,decreased range of motion, fixed joint deformity and soft tissuecontractures, increase the frequency and duration of remission ordisease-free periods and prevent or attenuate chronic progression of thedisease. In these embodiments, the glandular kallikrein composition isarranged to be injected directly into the afflicted joints or takenorally. Preparation of the glandular kallikrein composition forinjection is described herein.

Glandular kallikrein compositions could also be used to induce oraltolerance against graft rejection and sprayed or applied to tissuegrafts or organs prior to transplantation. As discussed above, theglandular kallikreins or bioactive fragments thereof induce toleranceand inhibit the immune response, meaning that prior oral application ofthe glandular kallikrein compositions would inhibit rejection of thetransplanted material. The glandular kallikrein composition willaccomplish at least one of the following: prolong the life of the graft;decrease the side effects associated with immunosuppressive therapy anddecrease accelerated atherosclerosis associated with transplants. Inother embodiments, a mesh coated or arranged to release the glandularkallikrein composition may be used in lieu of spray application.

Juvenile Diabetes or Type I Diabetes is a chronic condition in which thebeta cells in the pancreas make little or no insulin because these cellsare destroyed by the host immune system. This in turn destroys the isletcells' insulin-producing capacity which in turn brings on diabetes.Thus, the early administration of glandular kallikrein orally or abioactive fragment thereof, in combination with antigen(s) linked to orimplicated in Juvenile Diabetes will accomplish at least one of thefollowing: decrease severity of symptoms, decrease severity of clinicalsigns, increase time periods between insulin treatments, increase thefrequency and duration of remission or disease-free periods and preventor attenuate chronic progression of the disease.

The invention provides kits for carrying out the methods of theinvention. Accordingly, a variety of kits are provided. The kits may beused for any one or more of the following (and, accordingly, may containinstructions for any one or more of the following) uses: treatingmultiple sclerosis, myasthenia gravis, arthritis, inflammatory boweldiseases, tissue grafts, in an individual; preventing an autoimmuneresponse, inflammation or prolonged inflammatory response in anindividual at risk of multiple sclerosis, myasthenia gravis, arthritis,inflammatory bowel diseases, tissue grafts; preventing one or moresymptoms of an autoimmune response, swelling, pain, inflammation,prolonged inflammatory response or the like in an individual at risk ofmultiple sclerosis, myasthenia gravis, arthritis, inflammatory boweldiseases, tissue grafts; reducing severity one or more symptoms of anautoimmune response, swelling, pain, inflammation, or prolongedinflammatory response in an individual; reducing recurrence of one ormore symptoms of an autoimmune response, swelling, pain, inflammation,or prolonged inflammatory response in an individual; suppressing anautoimmune response, swelling, pain, inflammation, or prolongedinflammatory response in an individual at risk of multiple sclerosis,myasthenia gravis, arthritis, inflammatory bowel diseases, tissuegrafts; delaying development of an autoimmune response, swelling, pain,inflammation, or prolonged inflammatory response and/or a symptom ofmultiple sclerosis, myasthenia gravis, arthritis, inflammatory boweldiseases, tissue grafts in an individual; and reducing duration of anautoimmune response, swelling, pain, inflammation, or prolongedinflammatory response in an individual.

The kits of the invention comprise one or more containers comprising aglandular kallikrein or a bioactive fragment thereof, a suitableexcipient as described herein and a set of instructions, generallywritten instructions although electronic storage media (e.g., magneticdiskette or optical disk) containing instructions are also acceptable,relating to the use and dosage of the glandular kallikrein for theintended treatment (e.g., multiple sclerosis, myasthenia gravis,arthritis, inflammatory bowel diseases, tissue grafts, or the like). Theinstructions included with the kit generally include information as todosage, dosing schedule, and route of administration for the intendedtreatment. The containers of the glandular kallikrein may be unit doses,bulk packages (e.g., multi-dose packages) or sub-unit doses. Asdiscussed above, antigens for specific autoimmune disorders or allergiesmay be incorporated into the pharmaceutical composition or may beprovided as separate pharmaceutical compositions, prepared as describedabove. The kit may also include specific antigens for co-administrationwith the glandular kallikrein or may be incorporated into thepharmaceutical composition.

As discussed above, glandular kallikrein (GK) is an enzyme of the serineprotease family capable of generating biologically active peptides bypartially degrading various substrates. It is found in several tissueswith particularly high concentrations in salivary glands, pancreas,kidney and the prostate gland. The physiological functions of thisenzyme appear to vary according to the tissue in which it acts and thesubstrate(s) available in such tissues.

Several in vitro effects of kallikreins on cells of the immune systemhave been reported. Thus, several authors have described mitogenic andco-mitogenic effects of kallikrein and other serine proteases. Suchmitogenic effects were observed with thymocytes (103), T cells and Bcells (104). Although bradikinin may also have mitogenic effects (105),the involvement of this kinin in kallikrein-induced mitogenesis is notwell investigated. Moreover, several proteases, including kallikrein,were shown to be involved in immunoglobulin isotype control. ThusIshizaka described a kallikrein-like factor calledglycosylation-enhancing factor, which induced CD4+ T cells to produce anIgE-potentiating factor and to favour the production of IgE by memory Bcells (106). Serine proteases from Schistosoma mansoni schistosomulawere reported to enhance IgE production (107). Moreover, the addition ofkallikrein and other serine proteases in various concentrations tocultures of B cells stimulated with LPS and IL 4 enhanced the productionof IgE, IgG1, or IgG3, depending on the enzyme concentration used (108).

Our interest in GK arose from studies on immunosuppressive factors inthe SM gland of rats. The addition of crude extracts from rat SM glandsto murine spleen and lymph node cultures stimulated with concanavalin A(Con A) induced either suppression (at high concentrations) or furtherstimulation (at lower concentrations) of proliferative activity (109).This suggested that these extracts contained factors with suppressiveeffects as well as factors with the ability to enhance lymphocyteproliferation. Gel filtration of the crude extracts revealed that the invitro suppressive activity was due to factors with molecular weighthigher than 50 kilo-Daltons (kDa), while stimulation was due to factorswith molecular weight lower than 50 kDa (FIG. 4). We tested the in vivoactivity of both the higher and lower molecular weight fractions in theskin allograft, direct plaque forming cell response and in thedelayed-type hypersensitivity (DTH) models (110). As shown in Table 2,and contrary to what one might have expected in view of their in vitrosuppressive activity, the high molecular weight fractions did not haveany significant effect in these models. On the other hand, the lowermolecular weight fractions produced significant suppression in all threemodels.

Fractionation of the lower molecular weight pool of fractions throughthe successive steps of hydrophobic interaction, anion exchangechromatography and a final gel filtration step led to the isolation asingle protein (FIG. 5) which retained the properties of in vitrostimulation of lymphocyte proliferation and in vivo immunosuppression(results not shown). The isolated protein was amino acid sequenced (111)using an automated Edman degradation procedure (112). FIG. 6 shows thepartial N-terminal amino acid sequence of the 40 kDa protein and of themembers of the kallikrein family represented in the rat SMG. The x's(unidentified amino acids) in our sequence are probably cisteins whichare destroyed in the Edman degradation process. If this is taken intoaccount, the first 25 amino acids of our protein has identical sequencewith that of true GK and differ from those of other members of thekallikrein family. In view of the fact that no other rat proteins withthe same amino acid sequence are known, it was concluded that theisolated protein was true rat GK (rGK).

The esterase activity of the isolated rGK was about the same as that ofa commercially obtained porcine GK (pGK) when measured in the2-N-benzoyl-arginine ethyl ester (BAEE) assay (113). Differentconcentrations of aprotinin induced different degrees of inhibition.FIG. 7 demonstrates the effects of rGK and pGK in the presence or in theabsence of aprotinin on the proliferative activity of Con A stimulatedmurine lymph node cells. The same concentrations of pGK induced similarco-mitogenic effects. Con A concentration in these experiments was suchthat it induced only suboptimal mitogenic effects, suitable for thedemonstration of the co-mitogenic activity of rGK. The addition ofdifferent concentrations of aprotinin to the co-stimulated culturesinduced dose dependent suppression. It should be noted that the highestconcentration of aprotinin used in this experiment (1.5 μg/ml or 6μg/culture) was capable of inducing about 90% inhibition in the BAEEassay. On the other hand, the lowest concentration of aprotinin (1.5μg/culture) induced about 40% inhibition of the enzymatic activity andproduced partial inhibition of the co-mitogenic activity.

The results of an in vivo experiment along the same lines are presentedin FIG. 1. A DTH reaction was induced in mice sensitized with picrylchloride and challenged with the same agent six days later. Theinjection of rGK 24 hr before challenge resulted in an almost completesuppression of the response. Similar suppression was obtained with pGK.The dose of rGK used in this experiment (57 μg/animal) was based on ourprevious experience. The higher of the two aprotinin doses (190μg/animal) was selected so as to provide, after dilution in the bloodstream, a concentration similar to that used in the in vitroexperiments. The suppressive effects of rGK and pGK were almostcompletely removed by the injection of this higher dose of aprotiningiven immediately before GK injection. On the other hand, a lower doseof aprotinin induced only partial suppression of the rGK effects. Thesetwo experiments clearly demonstrate that the enzymatic activity of GKmust be preserved in order to retain its in vivo and in vitroimmunological effects.

Table 3 demonstrates the effects of varying the time of GK injectionwith respect to the time of immunization or of challenge in the DTHmodel in mice. If given before immunization, GK had a suppressive effectlasting for at least fourteen days. This suggested that the animals didnot develop any immunity when presented with the antigen. On the otherhand, if GK was given after the development of immunity, it induced ashort lived suppression of the skin reaction with a full recovery ofreactivity a week after GK administration. This demonstrated that GK didnot affect the state of immunity of an animal and suppressed the skinreaction with a mechanism that may be either immunological oranti-inflammatory. It should be noted that the half life of GK is suchthat 24 hours after injection, i.e. at the time of antigenadministration for either immunization or challenge, none of theinjected GK is present in the animal Thus, GK probably acted indirectly,via the mediation of some products of its enzymatic action.

FIG. 8 shows the results of an experiment in the collagen arthritis (CA)model in rats. A single injection of GK given at the time when thearthritic reaction begins to flare up induced an almost completesuppression lasting 4-5 days followed by the return to an almostunmodified arthritic reaction. On the other hand, repeated injectionsmaintained suppression for the entire duration of the experiment. Thus,this experiment confirmed that the effects of GK in immune animals areshort lived and do not reduce the state of immunity of the animals.

The effects described up to this point refer to in vitro phenomena or tothe subcutaneous administration of GK. The fact that GK is found in highconcentration in salivary glands and is secreted in saliva suggests thata significant physiological function of GK may occur via externalsalivary secretion.

For this reason, we also tested the effects of orally administered GK inrats. Experiments of this nature were carried out in rats using thecollagen arthritis model. This involves the injection of Type IIcollagen in an oil based adjuvant which induces an arthritic reactionbeginning two weeks after immunization and lasting for the next 3-4weeks. The variables we tested included the effects of a pre-treatmentwith oral collagen to induce tolerance to the subsequent immunization,the effects of the surgical removal of the submandibular gland and thoseof oral GK, given before tolerization and/or before immunization tonormal and, sialoadenectomized (SMGx) rats. FIG. 2 demonstrates theresults of one of such experiments. The oral pre-treatment with Type IIcollagen significantly reduced the arthritic response in normal rats butdid not have such effect in SMGx animals. On the other hand, the oraladministration of GK significantly reduced the arthritic reaction. FIG.8 shows the results of another experiment which confirmed thatsialoadenectomy interferes with tolerance induction. Moreover, thisexperiment demonstrated that the oral administration of GK to SMGx ratsrestores the ability of the mucosal immune system of these animals todevelop tolerance upon oral collagen administration.

These results point to similarities and differences in the action of GKdepending on whether it is used in vitro or in vivo and whether it isinjected or given orally. The in vitro effects consisted of thestimulation of lymphocyte proliferation, while the in vivo effectsappeared to be immunosuppressive. This apparent discrepancy may beexplained if the in vivo effects, rather than true suppression,represented some form of immune deviation involving the reduction of theresponses under investigation and the stimulation of other responses,not studied by us. The most likely mechanism for such an immunedeviation would be a decrease of T_(H1) activity and increase of T_(H3)suppressor/regulatory T cell action. The responses we found to besuppressed by GK treatment were cell-mediated immunity (DTH, allograftrejection and CA) or T-dependent IgM production (direct PFC response).These responses would be suppressed by any mechanisms that reduce T_(H),activity or favor the switch from IgM to any other immunoglobulin class.This explanation would be consistent with the fact that oral GK favorsthe induction of oral tolerance. This reaction is thought to involve adeviation from cell-mediated responses to IgA production induced byincreased activity of TGF-β producing T_(H3) cells. Alternatelysuppressor cells may be activated by GK. Yet another possibleexplanation for the apparent discrepancy of in vitro versus in vivoeffects would suggest that in vitro stimulation of lymphocyteproliferation was due to the formation of stimulatory peptides, formedby the action of GK on some substrate(s) contained in the culturesystem. Under in vivo conditions, the proteolytic action of GK result inthe generation of suppressor/regulatory T_(H3) lymphocytes. It is likelythat the in vitro proliferative response reflects the expansion of theseregulatory cells.

Differences in GK action were also observed when the route of GKadministration was changed from subcutaneous to oral. In bothsituations, some suppression of immune reactions was observed if thetreatment was simultaneous or shortly preceded antigen administration.On the other hand, the oral administration of GK appeared to enhance theinduction of tolerance if given together with an oral antigen. Theensuing suppression of the arthritic reaction exceeded in duration andin magnitude the “immunosuppressive” effect of GK alone administeredeither by injection or by mouth. The same experiments also demonstratedthat oral tolerance could not be induced by antigen alone insialoadenectomized animals. Oral tolerance is a state of antigenspecific hyporesponsiveness subsequent to the oral delivery of anantigen. It represents a protective reaction by the gut associatedmucosal tissue (GALT) to prevent unnecessary and potentially harmfulreactions to food antigens. It involves more than one mechanism. Highantigen doses induce clonal deletion, while lower doses induce an activeform of suppression or immune deviation, mediated by TGF-β producing Tcells, referred to by some authors as T_(H3) cells. The action of thesecells suppresses T_(H1) responses and favors T_(H2) responses (114-116).From Peyer's patches, these regulatory cells migrate to peripherallymphoid organs and to all other tissues and organs, thus renderingsystemic the immunosuppressive effect. Our results suggest that the SMgland plays a significant role in maintaining the normal responsivenessof GALT and that GK secretion in saliva is one of the factors involvedin this function. The immunological effects of GK make this molecule aninteresting candidate in the treatment of autoimmune diseases.

An immunosuppressive peptide of 40 kDa molecular weight, isolated fromthe submandibular glands, (SMG*) of rats, had the capacity to suppressimmune reactions upon parenteral administration to rats and mice. Thepeptide was identified as glandular kallikrein (K1) by partialsequencing and by enzymatic activity. Further experiments revealed thatthe excision of SMG (SMGx) from Lewis rats prevents the oral inductionof immunological tolerance against native bovine type II collagen(BCII). Thus SMGx rats, when they were given oral BCII and weresubsequently immunized with the same antigen, developed collagen inducedarthritis (CA), while normal rats given an identical treatment wereprotected from the disease. When SMGx rats were orally given porcine K1in conjunction with BCII, the capacity of such animals to develop oraltolerance was fully restored. These results indicate that normal SMGfunction is required for the induction of oral tolerance and that K1 isinvolved in mediating this function.

On the basis of current understanding of mucosal immunity and of theinduction of immunological tolerance by the oral/nasal route thisdiscovery will have relevance to the treatment of the following diseaseconditions: (i) autoimmune disease, for example, rheumatoid arthritis,multiple sclerosis, diabetes, myasthenia gravis, and the like. In thesecases it is anticipated that the oral administration of K1 jointly withantigen would lead to the induction of tolerance, which in turn wouldcause the amelioration of symptoms; (ii) Inflammatory conditions, forexample, colitis, connective tissue diseases and the like, in which asimilar strategy may be feasible if the antigen/irritant is known; (iii)gene therapy; (iv) finally it is possible that the effectiveness of oralvaccination could be increased by the simultaneous oral administrationof K1 inhibitors.

In one embodiment, there is provided a new approach for theamplification of orally induced immunological tolerance in patientssuffering from rheumatoid arthritis. This new treatment involves thejoint oral application of antigen and K1 of animal (eg. porcine) orhuman origin to patients. It is anticipated that K1 will increase theefficiency of oral tolerization with antigen, so that a new protocol fortherapy could be developed.

The submandibular gland of laboratory rodents has been known to be anendocrine organ for a long time. It is also integrated into theimmunoregulatory network Immunoregulatory cytokines, such as TGF-β andimmunoregulatory hormones, including nerve growth factor, epidermalgrowth factor, are produced in significant quantities by the SMG (1). Wehave identified glandular kallikrein as a powerful immunosuppressiveprinciple in rat SMG (2-5), as outlined below.

The experimental evidence obtained in our laboratories is the following:

1) Semi-purified extracts of the SMG exert a suppressive effect on theantibody response, on contact sensitivity reactions, allograft rejectionand on adjuvant-induced arthritis (2-5).

2) The active principle capable of mediating these effects was purifiedfrom rat SMG to homogeneity and shown to be a 40 kDa protein that waspartially sequenced and found to be identical with glandular kallikrein(rK1).

3) The 40 kDa rK1 had the characteristic esterase activity of K1. The invitro effects on lymphocyte proliferation and the in vivoimmunosuppressive activities of rK1 were dependent on preserving itsenzymatic activity.

4) Parenteral rK1 and porcine (p) K1 suppressed the contact sensitivityreaction to picryl chloride in mice. This suppression was inhibited whenthe animals were treated with the specific enzyme inhibitor, aprotininin addition to K1 (FIG. 1).

5) We also demonstrated that the surgical removal of the submandibulargland (SMGx) in Sprague Dawley rats decreased the severity ofcollagen-induced arthritis (CA) and prevented the induction of oraltolerance in animals fed with native type II bovine collagen. Moreover,treatment of Sprague Dawley rats s.c. with rK1 after immunization withBCII suppressed the development of arthritis (FIG. 2) These resultssuggest that the SMG exerts both a stimulatory and suppressive effect onthe autoimmune process.

6) In the collagen-induced arthritis model in Lewis rats, type II bovinecollagen (BCII) in oil, given intradermally induced an arthriticreaction, which reached its maximum 4-5 weeks later. In this model, theoral administration of BCII (3 μg/day) prior to the induction on days−7, −5 and −3, suppressed the development of arthritis. If thesubmandibular gland was removed, the rats could not be tolerized in thismanner. On the other hand, the oral administration of pKl (100 IU/day)given on days −8, −5, and −3, fully restored the oral induction ofimmunological tolerance. The animals so treated were thus protectedagainst the induction of arthritis by the subsequent challenge with BCII(FIG. 3, Table 1). Taken together, these experimental results indicatethat:

a) K1 is a powerful immunosuppressive agent, which is capable ofexerting a systemic effect when given orally or parenterally;

b) Collagen induced arthritis in rats can be treated by the parenteraladministration of rK1;

c) Salivary function is necessary for the induction of oralimmunological tolerance to CA; and

d) K1 restores the capacity to develop oral tolerance in animals withdeficient salivary functions.

Two main hypotheses may be suggested to explain these results.

-   Hypothesis 1. K1 induces changes of immune responsiveness that    include the suppression of ongoing immune responses and the    facilitation of oral tolerance induction. These changes could be due    to a direct action of K1 on immunocompetent cells or, more likely,    may be mediated by the activation of biologically active molecules    by the enzymatic action of K1. This effect could require doses    higher than physiological and may not be related to any    physiological function of salivary K1. The effect hypothesized here    could be defined as a pharmacological one. According to this    hypothesis, SMGx would produce alterations of the mucosal lymphoid    tissue that may be due to mechanisms other than the loss of K1    secretion. In this case, the oral administration of K1 would    overcome these effects in spite of the persisting deficiency of    other salivary factors.-   Hypothesis 2. Salivary K1 is a physiological regulator of mucosal    immunity. According to this hypothesis, SMGx would deprive the    animals of this essential factor and cause changes of the mucosal    immune system that prevent oral tolerance induction. In this case,    only the replacement of oral K1 would restore the normal balance in    the mucosal lymphoid tissue.

The distinction of these two possibilities is important if one wants topredict the possible therapeutic applications of oral K1 and antigen inautoimmune conditions. In the first hypothesis, K1 would be expected topotentiate oral tolerance induction, irrespective of whether thesalivary function is normal or not. In the second hypothesis, onlypatients with a deficient salivary secretion of K1 would benefit fromthe treatment.

Current evidence does not allow us to distinguish between these twopossibilities. Results demonstrating a strong suppression of ongoingimmune responses by parenteral application of K1 support the firsthypothesis (FIG. 1.) (2). On the other hand, the more recent resultswith the oral administration of K1 jointly with BCII in SMGx ratsappears to support the second hypothesis, while still being compatiblewith the first one (FIGS. 2,3). The two above hypotheses are notmutually exclusive. It would be totally consistent with our currentevidence to suggest that K1 may have a physiological role in maintaininga normal mucosal immune function while also inducing pharmacologicaleffects in doses larger than physiological. In this case, oral K1therapy would be beneficial in autoimmune conditions irrespective of anormal or deficient salivary function in the patient.

Lewis rats showed an excessive activation of the kallikrein-kinin systemduring experimental autoimmune inflammatory disease, whereas in Buffalorats, a self-limiting disease developed, which was associated with adecreased rate of kininogen cleavage (18). Transgenic miceover-expressing the rat kallikrein binding protein (RKBP) showedelevated resistance to the lethal effect of endotoxin (19). Theexpression of human tissue kallikrein genes in mice had a profoundeffect on the histological structure of lymphoid tissue and led to ageneral decrease of lymphocytes, particularly in T-cell dependent areas(20). Immunoreactive K has been co-localized with prolactin in pituitaryadenomas, where a role in processing of PRL to its 22 kDa form has beensuggested. It has also been detected in normal pituitary tissue (9, 21).

In general kallikreins are regarded as important local(autocrine-paracrine) regulators, that fine tune blood supply toinflamed tissues; stimulate the release of prolactin and growth hormonefrom the pituitary gland (which have a pro-inflammatory effect);increase CI and glucose transport; participate in the generation of painsensation; release transmitters from neurons; and stimulate DNAsynthesis and growth in at least some cells, such as osteoclasts andendometrial stromal cells (9).

-   (ii) Kallikrein in rheumatoid arthritis. K has been detected in    synovial fluid from arthritic joints (22), bronchial lavage from    asthmatics (23) and in nasal secretions of patients with rhinitis    (24, 25). The source of GK in these inflammatory conditions is    thought to be nasal and pulmonary secretory glands and infiltrating    neutrophils (26).

Several studies ranging from the 1960s to very recent ones have shownthat a significant proportion of patients suffering from rheumatoidarthritis (RA) have deficient salivary secretion (27, 30). Thisdeficient secretion includes not only a relatively small number of RApatients with secondary Sjögren's Syndrome (SS) but also a larger groupof patients with sicca syndrome who are not usually classified as havingSS. The reported proportion of patients with deficient salivary outputvaries from 30% to about 65%, with most of the authors giving figures inthe 40-50% range.

Unfortunately, most of the authors who have studied salivary kallikreinsecretion in RA patients report salivary concentrations only and thetotal output has not been calculated (31, 33). If one takes into accountthat flows are sometimes markedly reduced (up to 90% reduction—34), thismay result in a significantly decreased K1 secretion even if it ispresent in the saliva at slightly increased concentrations. Friberg etal., (35) found that the salivary concentration of K in 7 patients withSS and no steroid treatment was 808+/−179 units/liter (U/L;mean+/−S.D.). Stimulated whole morning saliva output for these patientshas been 0.35+/−0.18 ml/minute. The total output of K for this group ofpatients is 0.283 U/min. In the second group of 7 SS patients treatedwith steroids the salivary concentration of K was 361+/−265 U/L and thesecretion rate 0.32+/−0.29 ml/min. Total output: 0.116 U/min. Kconcentration in saliva of 9 normal individuals was 170+/−83 U/L andsaliva secretion 1.77 ml/min. Total production of K in normalindividuals was 0.301 U/min. It is evident from these results that theincreased K concentration in the saliva of the first group of patients,coupled with a major reduction of saliva production resulted in nearnormal output. On the other hand, total K production of SS patients thatwere on steroids was 38% of normal -clearly deficient. The mean totaloutput of all patients was 0.199 U/min, which amounts to 66% of controlvalues. It is worth noting that the group receiving no steroids wasmainly composed of primary SS patients (without RA), while the groupreceiving steroids consisted mainly of RA patients with secondary SS.Recent findings suggest that blocking autoantibodies to theacetylcholine receptor present in the serum of primary Sjogren'ssyndrome can cause secretary abnormalities (36). This could explain theelevated K in the saliva of such patients as fluid and electrolytesecretion by the major salivary glands is under cholinergic control,whereas protein secretion is regulated by sympathetic nerve fibers thatoriginate in the superior cervical ganglion (1). In the absence offurther data, it cannot be established whether the low GK output in thesecond group was solely due to the use of steroids or also to thepresence of RA.

Matthews et al. (37) reported a significant reduction of salivary flowin a group of 20 RA patients compared with 20 controls. Parotid flow inRA patients was 0.086 ml/minute, versus 0.196 ml/min in the controls(p<0.002). Submandibular flow in the RA group was 0.114 ml/min, versus0.186 ml/min in the controls (not significant). Kallikreinconcentrations were only, and non-significantly, reduced with averageconcentrations of 0.304 units/mg of protein for parotid saliva (versus0.557 in the controls) and 0.308 units/ml in submandibular saliva(versus 0.463 in the controls). Protein concentrations were practicallyunchanged. From these data, one may conclude that total GK output wasreduced in the RA group. This study reports only data for the entiregroup of patients. Thus, it probably underestimates the reduction thatmust have taken place in the subgroup of patients complaining ofxerostomia (15 out 20 patients).

-   (ii) Oral immunological tolerance is a well known phenomenon (first    described in 1912) that is currently receiving much attention as a    means to treat autoimmune diseases (56). There appear to be    mechanisms for this phenomenon depending on the dose of the antigen.    High antigen doses induce deletion or anergy of specific clones of    T_(H1) and T_(H2) cells, while low doses induce the emergence of    T_(H2) clones that produce IL 4 and IL 10 and of T_(H3) clones that    produce TGFβ. These, in turn, suppress responses mediated by T_(H1)    cells. Thus, high doses of antigen appear to induce a true state of    tolerance, while the effects of low doses would be better described    as immune deviation phenomena. The successes of oral tolerization in    experimental animals suggested its use in human autoimmune diseases.    Several clinical trials have been performed in autoimmune diseases    (56). Of particular interest here are the results of four trials    (57-61) in RA patients. Such results demonstrated that RA patients    receiving low doses of collagen showed statistically significant    improvements. However, such improvements were not good enough for    oral tolerance to become right now a commonly practiced therapy in    RA. Some of the possible ways considered by various authors (60, 63)    to improve the effectiveness of oral tolerance are: (i) better    selection of patients; (ii) better selection of doses; and (iii) the    use of additional treatment with adjuvants in order to potentiate    tolerance.

As will be well known by one of skill in the art, gene therapy involvesthe insertion of non-host DNA into host cells, typically by viralvectors. A fundamental aspect of this technology is that the host mustbe tolerant to the virus used. Thus, another potential use of theabove-described glandular kallikrein preparations would be to inducetolerance to viral vectors for gene therapy by administering theglandular kallikrein as described above when administering a viralvector for gene therapy using means known in the art. It is of note thatthis method would be suitable to induce such tolerance, which is apotentially important application.

As discussed above, described herein are methods of inducing toleranceto “foreign” material by administering glandular kallikrein as a methodof treating or ameliorating for example autoimmune diseases.Specifically, as discussed above, the glandular kallikrein preparationsare used to induce tolerance to the “foreign” material. It is of notethat depletion of K1 will in fact decrease tolerance and in turn favoursimmunization, meaning that depletion of glandular kallikrein could beused for the potentiation of oral vaccines. Here it is envisioned, thatthe oral administration of an K1 inhibitor, for example, aprotinin in anamount effective to inhibit or reduce the activity of the glandularkallikrein with the antigen would be required to achieve an adjuvanteffect.

While the preferred embodiments of the invention have been describedabove, it will be recognized and understood that various modificationsmay be made therein, and the appended claims are intended to cover allsuch modifications which may fall within the spirit and scope of theinvention.

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TABLE 1 Functional disability in rats with arthritis. Days afterchallenge 14 16 18 21 *23 25 28 31 35 38 42 1. Immune 0/4 0/4 0/4 0/42/3 2/3 2/3 2/3 2/3 2/3 2/3 2. Imm + Tol 0/5 0/5 0/5 0/5 0/4 0/4 0/4 0/40/4 0/4 1/4 3. Imm + SMX 0/5 0/5 0/5 0/5 1/4 2/4 2/4 2/4 2/4 2/4 2/4 4.Imm. + Tol + 0/5 0/5 0/5 0/5 1/4 2/4 2/4 2/4 3/4 2/4 2/4 SMX 5. Imm. +Tol + 0/5 0/5 0/5 0/5 0/4 0/4 0/4 0/4 0/4 0/4 0/4 SMX + pK1 The animalswith functional deficit and deformities in their paws/total number ofanimals in the groupare are given. *On day 23 one animal was killed fromeach group for histologic examination.

TABLE 2 Effects of high molecular weight (HMW) and low molecular weight(LMW) pools of gel filtration fractions in three in vivo immunologicalassays. Modified from Ref. 110. Exp. Model Treatment^(a) Groups Results± SD^(b) Significance^(c) Skin 10 daily doses PBS 12.2 ± 0.37transplantation CBA/2J to Days 0 to 9 HMW 13.0 ± 0.44 NS C57B1/6J LMW14.7 ± 0.76 p < 0.05 Direct PFC  5 daily doses PBS 237.0 ± 19.7  Days 1to 3 HMW 193.0 ± 12.0  NS LMW 119.6 ± 10.0  p < 0.05 DTH  2 daily dosesPBS 19.0 ± 0.70 (A/J mice) Days 4 and 6 HMW 18.2 ± 0.49 NS LMW  9.0 ±1.00 p < 0.01 ^(a)The animals received the subcutaneous injection of 200μl of PBS or PBS containing either the HMW or the LMW fractions. Thedoses corresponded to one-half of a SM gland (0.965 mg or 0.53 mg,respectively) and to one gland in the other models (1.93 mg or 1.06 mg,respectively). ^(b)skin transplantation results are expressed as meansurvival time; PFC response is expressed as the number of IgM anti-SRBCPFC per one million splenocytes; DTH results are expressed as increasesof ear thickness in 1/10 mm units 24 hours after challenge.^(c)Significance was determined by two-sample t-test, using thePBS-treated group as the control; NS, not significant.

TABLE 3 Effects of the intradermal injection of rGK given before orafter immunization in the DTH model. Test on day: b Treatment^(a) 7 14None 18.4 ± 0.76 17.6 ± 0.92 rGK, Day −1  6.0 ± 1.2*  7.6 ± 0.98* rGK,Day +6  4.4 ± 0.63* 16.8 ± 1.12 rGK, Day +13 19.0 ± 1.24  5.6 ± 0.78*^(a)A/J mice were immunized by the application of 0.1 ml of a 5%solution of picryl chloride in ethanol to the skin of the abdomen; theday of immunization is referred to as day 0; the animals a single doseof 60 μg of rGK in 200 μl of PBS on the days indicated. b. Animals weretested with the application of a 1% solution of picryl chloride in oliveoil on one ear. Results are expressed in terms of the increase if thechallenged ear over the pre-challenge values using units equivalent to1/10 mm. *p < 0.05.

1. A method of suppressing an immune reaction to beta cells in a humanafflicted with or exhibiting at least one clinical sign of type 1diabetes, comprising administering to the human about 10 to about 5,000International Units of a glandular kallikrein, and thereby suppressingthe immune reaction to beta cells.
 2. The method of claim 1, wherein theglandular kallikrein comprises kallikrein-1, a fragment of a kallikreinhaving serine protease activity, or a mixture thereof.
 3. The method ofclaim 1 or 2, wherein the glandular kallikrein is administered orally.4. The method of claim 1 or 2, wherein the glandular kallikrein isadministered parenterally.
 5. The method of claim 1 or 2, comprisingadministering the glandular kallikrein and a beta cell antigen.
 6. Themethod of claim 5, comprising co-administering the beta cell antigenwith the glandular kallikrein.
 7. The method of claim 6, wherein theglandular kallikrein and the beta cell antigen are administered orally.